Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology

Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 1

Nitrated Fatty AcidsFormation and Actions

Bruce Freeman, PhDDepartment of Pharmacology

University of Pittsburgh School of Medicine


Objectives of Course Lecture and Supplied Materials

• Discuss the biological formation and structural characterisitics of nitrated fatty acid derivatives

• Teach how to synthesize, purify, quantify and handle nitrated fatty acids

• Convey present knowledge about the cell signaling actions of nitrated fatty acids


Fatty AcidsFatty acids have common names, IUPAC names and acronyms

Linoleic AcidOctadeca-9,12-dienoic acid18:2 LA

Arachidonic AcidEicosatetraenoic AcidDodeca-5,8,11,14-tetraenoic acid20:4AA

O

HO

Myristic Acid (14:0) Linolenic Acid (18:3) Palmitic Acid (16:0) Arachidic Acid (20:0) Stearic Acid (18:0) Docosahexanoic acid (22:6) Oleic Acid (18:1)

OH

O

Endogenous fatty acids have the cis-stereochemical orientation


• NO manifests both pro-oxidant and antioxidant actions

• Reactions of NO-derived species with oxidizing unsaturated fatty acids yields NO2 derivatives

LNO2 Formation

J Biol Chem 269:28066-28075, 1994

A Crucial Experiment


Species Compartment Concentration (nM)

NO-2 Plasma

RSNO

Plasma

LNO2 Plasma

Free

Esterified

Total

Hb-NO Blood < 50

Hb-SNO

Blood

< 50

LNO2

Packed red cells Free Esterified

Total

LNO2 Blood

205 +_ 217.2 +_ 1.1

79 +_ 35

550 +_ 275 630 +_ 240

50 + 17_ 199 +_ 121

477 +_ 128*Total

TotalTotal

TotalTotal

Fraction

3-Nitro-tyrosine Plasma 0.7 +_ 0.3 Total

249 +_ 104

OANO2

Plasma

Free Esterified

Total

619 +_ 52

302 +_ 369 921 +_ 421

ONO2

Packed red cells Free

Esterified Total

59 + 11_ 155 +_ 65 214 +_ 76

OANO2

Blood

639 +_ 366*

Total

Nitrated Fatty Acids – Most Abundant BioactiveOxides of Nitrogen in the Vascular Compartment

PNAS 101:11577-11582, 2004 J Biol Chem, 2005


16:1

18:1

18:2

18:3

20:4

20:5

-NO2-(OH)NO2

Time (min)5 10 15 5 10 15

Plasma Urine

Time (min)

Bas

e P

eak

Inte

nsi

ty

5 10 15 5 10 15

-NO2-(OH)NO2

All Unsaturated Fatty Acid Species Display NitratedDerivatives In Healthy Human Plasma and Urine

J Biol Chem, in press 2005


Inflammatory Conditions Induce Oleic Acid Nitration Fatty Acid Micellar Emulsion

Control

NO2-, pH 3.0

MPO/H2O2/ NO2-

ONOO-

0.12

0.76

1.45

3.31

Condition NO2-oleic acid (nM)

MPO- 50 nM, H2O2- 600 µM, NO2-- 100 µM, ONOO-- 1.5 mM

Linoleic acid nitration results can be found in: O’Donnell et al.

Chem Res Tox 12: 83-92, 1999.

* p< 0.05

*

*

*

J Biol Chem, in press 2005


Inflammatory Conditions Induce Fatty Acid Nitration Intraperitoneal Injection of LPS in Mice

* *

* *

* p< 0.05, 3-4 animals

*

* *

*

Condition NO2-oleic acid NO2-linoleic acid

Heart (nmol/g)

Control

LPS

Lung (nmol/g)

Control

LPS

Plasma (nM)

Control

LPS

Kidney (nmol/g)

Control

LPS

38

323

0.017

0.066

0.017

0.060

0.030

0.215

29

120

0.006

0.030

0.014

0.044

0.009

0.069


Inflammatory Conditions Induce Fatty Acid Nitration Acute Respiratory Distress Syndrome Patients

* p< 0.05

Condition NO2-oleic acid NO2-linoleic acid

Matched plasma (nmol/mg prot) - 3.0 ± 0.7

Pulmonary edema fluid - 45 ± 6*


Mechanisms of Fatty Acid Nitration

• Acidic nitration• Enzymatic?

• Dietary sources

NO2+

NO2O2N

O2HOO

H H

O2N

NO2

(Nitronium Ion)Hydrogen

Abstraction


Why Were Nitrated Unsaturated Fatty Acids (Nitroalkenes) Not Identified and Studied Sooner?

• First identified in 1912 (Zelinsky), 1991 (Finlayson-Pitts)• To observe clinically-occurring nitrated fatty acids,

required development of sensitive MS capabilities • Decay rapidly with traditional lipid extraction and FA

derivatization techniques used for GC-MS

• NO2 group leaves peptides as m/z = 45 in positive ion mode, leaves NO2-FA adducts as m/z = 47

• NO2-FA adducts of proteins “lost” with typical thiol-based protein reduction and electrophoresis strategies

• NO2-FA adducts of proteins are retained on column with typical acetonitrile gradients used for peptide separation


Synthesis of Nitrated Linoleic Acid

PNAS 101:11577-11582, 2004


Detection/Characterization of LNO2

Method Results Comments

GC MS (EI)Structural data from standard

1. Very low sensitivity2. Derivatization

GC MS (NICI)In vivo detection

of LNO2

1. Good sensitivity2. Extensive sample

preparation3. Sample degradation

3-D Ion Trap ESI MS/MS

Structural data from standards

1. Low sensitivity2. MS3 and above,

not realistic

→ Unable to characterize/quantitate in vivo samples


Triple Quadrupole MS

Q1 Q2 Q3

CollisionChamberSource Detector

• MS – Scan specific mass range in either Q1 or Q3

• Product Ion Analysis – Set Q1 for specific mass; Q3 for product range

• Precursor Ion Analysis – Set Q3 for a specific product mass; Q1 for precursor range

• Multiple Reaction Monitoring (MRM) – Set Q1 for a specific precursor; set Q3 for a specific product


10

Peak 1 Peak 2

12 14

Time (min)10

Peak 1 Peak 2

12 14Time (min)

10

Peak 1 Peak 2

12 14Time (min)

Synthetic LNO2 Red Cell Plasma

HPLC Elution Profile for LNO2 Regioisomers

MRM transition: 324 [M-H]- / 277 [M-HNO2]-

-O

O NO2

-O

O O2NPeak 1

Peak 2

PNAS 101:11577-11582, 2004


157

171

196 233244

277

293

306324

x 7

150 200 250 300m/z

x 10

150 200 250 300m/z

Rel

ativ

e In

tens

ity

196171

277

244

293

306

233157157

171196

233244

277

293

306324

x 5

150 200 250 300m/z

277196

Standard Red Blood Cell Plasma

O

-O

NO2

10

Peak 1 Peak 2

12 14

Time (min)

Product Ion Analysis of LNO2

Regioisomers Peak 1PNAS 101:11577-11582, 2004


x 19

150 200 250 300

m/z

Rel

ativ

e In

tens

ity

277

244233

168

306324

228 168

x 7

228233

244

277

306

324

150 200 250 300

m/z

168

228

233

244

277

306

324x 5

m/z

150 200 250 300

277

228

Standard Red Blood Cell Plasma

O

-O

NO2

10

Peak 1Peak 2

12 14

Time (min)

Product Ion Analysis of LNO2

Regioisomers Peak 2 PNAS 101:11577-11582, 2004


PlasmaOA-NO2

Red CellOA-NO2

279

326

308

246

168

197

5X

279

326

308

246

168

197

5X

120 160 200240 280320m/z

SyntheticOA-NO2

279

326

308246168197

Synthetic[C]OA-NO13

2

120 160 200240 280320m/z

295

344

326308

2642X

9 10

9 10

9 10

9 10

time (min)

time (min)J Biol Chem, in press 2005

Nitrated Oleic Acid (OA-NO2) is Also Present in Human Blood


MS-Based Quantitation of NO2-FA

• Include [13C]-fatty acid and [15N]-nitrite during sample processing and analysis to detect artifactual fatty acid nitration

• Evaluate ranges of pH and adventitious NO2-

• Evaluate impact of oxidized lipid derivatives

• Use [13C]LNO2 and [13C]NO2-OA as internal standards to correct for sample preparation loss

• Internal standard curve linear over 5-orders of magnitude; limit of quantitation = 300 amol on column


1 2 3 4 5 6 7

324/277 (LNO2)

342/295 (13C-LNO2)

1010.10.010.001

5005050.50.05

10

1

0.1

0.01

0.001

[LNO2]/[IS]

ng/ml LNO2

y = 0.969x + 0.002r = 0.998

LOQ ~ 300 amol on columnStandard curve linear over 5-orders of magnitude

Quantitative Analysis of LNO2 in Clinical Samples

LNO

2 p

eak

are

aIS

pea

k a

rea

PNAS 101:11577-11582, 2004


Complex Lipid Nitro-Fatty Acid DerivativesMembrane and Lipoprotein Reservoirs?

“Stable” LNO2 StoragePLA2

-O

O NO2

O PO

OO -

N +LNO2

Palmitate

Cell SignalingNO2

Linoleic acid represents ~10% of net fatty acids in cells, with ~0.1-1.0% of net linoleate nitrated

~10-100,000 molecules LNO2/cell

NO 2

NO2

NO2NO2

NO2

NO2 NO2


0 2 4 6 8

0

400

800

1200

1600 OA-NO2

LNO2

+ Lipase

- Lipase

Time (hr)

Rel

ease

d F

atty

Aci

d (

nM

)

Lipase-Treated Complex Biological Lipids Release Nitrated Fatty Acids


+Prec (184.20): Exp 1, 10.761 to 29.474 min from Sample 1 (PC Mix) of Chromatography Test.wif... Max. 3.3e5 cps.

600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900m/z, amu

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

Inte

ns

ity, c

ps

758.8

782.8

786.8760.8

703.8

813.8

780.8788.8 815.8

756.8 816.8734.8762.8731.8 810.8705.8

735.8 806.8744.8675.8 802.8770.8 841.8720.8 846.8691.8 875.0 899.0650.6604.8 636.8622.8 695.6

Precursor scan m/z 184—molecular species of phosphatidylcholine

O PO

OO -

N +18:2

16:0

O PO

OO -

N +20:4

16:0


+Prec (184.20): 2.178 to 7.555 min from Sample 1 (PC + nitrated PC) of Precursor 184.wiff (Turb... Max. 2.9e5 cps.

770 775 780 785 790 795 800 805 810 815 820 825 830m/z, amu

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

3500.00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

1.00e4

Inte

ns

ity, c

ps

803.8

804.8

821.8

814.8

790.8788.8 798.8774.8 805.8 822.8819.8772.8 815.8812.8830.8791.8789.8 799.8775.8 828.8786.8780.8 801.8773.8 817.6807.8 818.6794.6 811.8770.8 824.8 827.8

Precursor Analysis of Nitrated Phosphatidylcholine (m/z 184)

O PO

OO -

N +LNO2

Palmitate


-EPI (787.50) CE (-45): 0.227 to 0.692 min from Sample 8 (EPI of 787) of nitrated PC.wiff (Turbo ... Max. 7.2e4 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z, amu

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

Inte

ns

ity, c

ps

787.5

255.2

480.3

741.5306.2

277.4 324.2549.3 658.6462.2 644.4 701.4 769.4757.5

EPI Analysis of Nitrated PC

Palmitate

LNO2

M[-CH3]


Nitroalkene Derivatives Redox-derived fatty acid derivatives that

display unique signaling reactivities

• Acidic hydrogen on C-N bond of nitroalkene capable of acid-base chemistry (Nef reaction), leading to NO release and cGMP-dependent signaling

•In “native” form, function as high affinity ligands for the nuclear receptor family PPAR

• C-N bond undergoes Michael addition reactions with nucleophiles (Cys, His)


Agilent Array Analysis of Cell Gene Expression Responses to LNO2

Human Monocytes, Endothelium

• Transcription and initiation factors

• Adhesion molecules, cytokines

• Enzymes, receptors

HO-1, PPAR-linked genesNitrolinoleic acid regulates 5504 genes

Up 2376 genesDown 3128 genes

Linoleic acid regulates 14 genes – 5 up, 9 down


NO Release From NitroalkenesVia Modified Nef Reaction

JBC 280:19289-19297, 2005


100

50

0

LNO2 LNO2

+ODQ

LNO2

+OxyHb

*

*

1010.10.01

0

20

40

60

80

100

LNO2 (µM)

(-) Endothelium

(+ ) Endothelium

L-NAME (30 µM)

Linoleate

Methanol

% R

elax

atio

n

% R

ever

sal R

elax

atio

n

Nitrolinoleate Mediates NO-Like Vascular Signaling

PNAS 99:15941-46, 2002

0 0.3 1 SNP (10 µM)0

2

4

6

cGM

P (

pm

ol/

mg

cel

l p

rote

in)

LNO2 (µM)


Nitroalkenes are Hydrophobically StabilizedOctyl-thio--glucopyranoside CMC=2.8 mg/mlOctyl--glucopyranoside CMC=7.3 mg/ml

[NO

] (µ

M/h

r)

EPR oxyMb

JBC 280:19289-19297, 2005


NO + 2o reaction products

K

k

EsterificationK

InflammationPLA2

PPAR activation

NO2

HO

O NO2

Kd

Kdk

HO

O NO2

NO2NO2

NO2

NO2

NO2

NO2

NO2

NO2

Hydrophobic Stabilization of LNO2

JBC 280:19289-19297, 2005


Nucleus

Cytoplasm

AGGTCA (N) AGGTCA

Target genesRXR

PPRE

PPAR

Coactivator

Co-repressor

Degradation

PPARLigands

P

u u

9-cis-RA

PPAR-Regulated Gene Expression


Nitroalkenes Induce PPAR-Dependent Cell Responses

• Induce CD36 expression• Inhibit TNF, IL-1, MCP expression, NFB• Inhibit vascular adhesion

Monocytes

Adipocytes• Induction of adipogenesis• Induce PPAR and aP2 expression• Increased glucose uptake Pulmonary Endothelium• Inhibition of TNF-induced VCAM-1 expression

Alveolar Epithelium• Lipid metabolism, apoptosis, redox signaling


Reporter Constructs for AnalyzingPPAR Ligand Activity

Nuclear receptor LBDs (ligand binding domain)

Gal 4 DBD (DNA binding domain)

Luciferase Luciferase activity

4 X Gal4

Full length PPARs

Luciferase Luciferase activity

3 X PPRE

Normalized to GFPCV-1 cells


LNO2 is a Specific PPAR Agonist

0

2

4

6

8

Vector

Control

LNO2

+ - + -+ -+ -

+- +- +- +-

*

*

*

Rel

ativ

e L

uci

fera

se A

ctiv

ity

PPARs

LNO2 Concentration (µM)

1

3

5

7

0 0.01 0.1 1 10

PPAR

PPAR PPAR

*

* *

Re

lati

ve

L

uc

ife

ras

e A

cti

vit

y

AR GR MR PR RXR

+ -+ -+ -+ -+ -

+-+- +- +- +-

Other nuclear receptorsPNAS 102:2340-2345, 2005


0 20 40 60 80 100 120Time (min)

100

80

60

40

20

01

1.5

2

2.5

3

0 0.2 0.4 0.6 0.8 1

Concentration (NO2-OA)

PPAR

PPAR

PPAR

0

3

6

9

NO2-OA(1 µM)

LNO2

(3 µM)Meth

LNO2

NO2-OA

Rel

ati

ve

Lu

cife

ras

e A

ctiv

ity

Rel

ati

ve

Lu

cife

ras

e A

ctiv

ity

Rem

ain

ing

nit

rate

d f

atty

aci

d (

%)

NO2-OA > LNO2 “potency” may be due to stability

NO2-OA is a More Potent PPAR LigandHO-1 expressionInhibition of VCAM-1 expressionAdipocyte differentiation, glc uptake

PNAS 102:2340-2345, 2005


Nitroalkenes Undergo Reversible Michael Addition Reactions with Nucleophiles - Thiols

N+O

O-

R'

H

H

RS- N+O-

O-

R'

H

H

RS

N+O-

O-

R'

H

H

RS

H+

N+O-

O-

R'

H

H

RS

H

200 300 400 500 600

m/z

0

25

50

75

100

Rel

ativ

e A

bu

nd

ance

306.3

324.2

631.3

GSH

LNO2

LNO2-GSH

Submitted

Nitroalkylation


Protein Nitroalkylation

4426 32 38Mass (103 m/z)

PurifiedGAPDH

GAPDH+

NO2-OA

GAPDHNO2-OA

100M GSH

MALDI-TOF-MS

2-D Ion Trap

327

327

327

327

Submitted


NO and Fatty Acid Signaling Converge

• NO terminates lipid radicals, inhibiting propagation reactions and preserving lipophilic antioxidants

• NO-derived species either stimulate or inhibit lipid and lipoprotein oxidation, depending on basal oxidative and inflammatory conditions

• NO is consumed by reactions of eicosanoid biosynthesis, gene expression and catalytic activity of eicosanoid biosynthetic enzymes regulated by NO

• Nitrated fatty acids transduce NO and NO2- signaling

formed by reactions of NO and NO2- derived

speciescan be misidentified as other NOx derivatives

signal via receptor-dependent mechanisms and unique chemical reactivities


Eugene Chen, PhD – MSMBruce Branchaud, PhD - UO

Date post:	12-Jan-2016
Category:	Documents
Upload:	shadow
View:	29 times
Download:	0 times

Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology

Documents